Use of primers for universal fingerprint analysis

ABSTRACT

The invention relates to the use of primers or primer pairs for DNA fingerprint analysis, wherein with the primers or primer pairs fingerprints are obtainable from humans as well as from animals as well as from plants as well as from microorganisms. The invention further relates to primers or primer pairs for the above-mentioned use.

This application is the national phase under 35 U.S.C. § 371 of priorPCT International Application No. PCT/EP97/00442, which has anInternational filing date of Jan. 31, 1997 and which designated theUnited States of America, the entire contents of which are herebyincorporated by reference.

The invention relates to the use of primers or primer pairs for DNAfingerprint analysis, wherein the use of the primers or primer pairsallows to obtain fingerprints from humans as well as from animals aswell as from plants as well as from microorganisms. The inventionfurther relates to primers or primer pairs for the above-mentioned use.

It is generally known that the presence of polymorphic andheterogenously dispersed repetitive sequences such as microsatellites isused for genetic analysis.

It is also well-known that retrotransposons such as copia elements ofDrosophila and copia-like elements in other species of the animal andplant kingdom usually are present as multiple copies in the genomes.Repetitive genomic sequences of this type were used in the example ofcopia-like elements in pisum (pea) for the genetic analysis of thisplant species (Lee et al., Plant Mol. Biol. 15 (1990), 707-722). Thismethod designated OFLP is based on a copia-specific primer and a secondprimer of a sequence of the retrotransposon flanking pea genome for PCRamplification. This made it possible to amplify pea varieties by PCRamplification of specific elements of the pea copia family and to testfor polymorphisms by separation of the non-radioactively labeled PCRproducts on an agarose gel and determine genetic relatedness. Also otherretrotransposons, e.g. Tos1-1, Tos2-1 and Tos3-1 from rice have beenused as molecular genetic markers for differentiation and identificationof rice cultivars by RFLP-analysis (Fukuchi et al., Jap. J. Genetics 68(1993), 195-204), while, however, also here it has been postulated thatfor other plant species their endogenous retrotransposons are isolatedfor use as a molecular marker. Another work (Purugganan and Wessler,Mol. Ecology 4 (1995), 265-269) uses a PCR-based method, which utilizesthe variation of restriction sites for restriction enzymes intransposable elements for a fingerprint analysis. All these methodsdescribed in the prior art have, however, in common that the describedgenetic markers or primers cannot be universally used in humans, plants,animals or microorganisms. It is obvious that the provision of suchgenetic markers or primers would offer essential advantages in manyareas of modern biology or medicine.

Thus, the problem underlying the present invention was to overcome theabove-mentioned drawbacks of the prior art and to provide methods andmeans which allow for a maximum degree of universal applicability of aminimum amount of primers or genetic markers for a fingerprint analysisof species from the plant and animal kingdom as well as of humans.

The solution to this problem is provided by the embodimentscharacterized in the claims.

It has surprisingly been found that the primers which hybridize withcopia-like elements in coconut (Cocos nucifera L.) and which in thissystem permit a fingerprint analysis can also be successfully used inmany other species of the animal and plant kingdom as well as in humansand even in microorganisms including yeast. This finding permits touniversally use said primers for fingerprint analysis in the wholeanimal and plant kingdom as well as in humans and in microorganisms.

Thus, the present invention relates to the use of a primer or primerpair for DNA fingerprint analysis, characterized in that the primer orprimer pair permits a fingerprint from humans as well as from animalsand plants as well as from microorganisms, wherein the primer or primerpair hybridizes with a DNA which is comprised in the copia-like elementof coconut (Cocos nucifera L.) as represented in FIG. 2B.

In this connection, the surprising results of the present invention areachieved with arbitrary combinations of different primers of oppositepolarity with the only requirement that they hybridize with thecopia-like element represented in FIG. 2B, as well as with the use of asingle primer which, due to the repetition of the copia-like elements,albeit in 5′→3′/3′→5′ orientation of two adjacent elements and not, asrepresented in FIG. 2B, in 5′→3′/5′→3′ orientation, likewise providesthe highly polymorphic fingerprints. It is self-evident that theaforementioned definition for the primers includes that they alsohybridize to DNAs from other organisms as long as they contain DNAsequences which correspond to DNA sequences from the above-mentionedcopia-like element.

The requirements for hybridization of the primer and subsequentamplification is derivable to the person skilled in the art withoutinventive effort from the prior art and the following examples.

The primers of the invention are preferably 15 to 25 nucleotides inlength. The invention, however, can also be carried out with primerswhich are shorter or longer.

The present finding is even more surprising since as a rule the priorart started from the assumption that primers can only be used forreliable fingerprints in taxonomically narrow limits.

In the prior art Rohde et al. (J. Genet. & Breed. 46 (1992), 391-394) itis described that highly repetitive sequences having homology to copiaelements described in other species exist in the genome of coconut(Cocos nucifera L.), which sequences are visible as two DNA bands, 1.3and 1.4 kilobases in length, respectively, after restriction of isolatedgenomic DNA with the restriction enzyme EcoRI and separation on anagarose gel. Three of these “Ecorep”-designated DNA fragments weresequenced after subcloning and sequence deviations could be determined.Attempts to use these differences for the genetic analysis of differentcoconut types by use of Ecorep sequences as a molecular probe in RFLPanalysis or by sequence specific PCR primers were not successful (Rohdeet al., J. Genet. & Breed. 46 (1992), 391-394; Rohde in: “La RechercheEuropeene au Service du Cocotier—Actes du Seminaire—8-10 septembre 1993,Montpellier”. CIRAD (Collection: Colloques du CIRAD), Montpellier, pages41-52).

Recently it was found for three coconut types that subfamilies of these1.3 and 1.4 kilobase Ecorep sequences exist, in which these elements areclustered in the coconut genome, i.e. they represent tandem repeats, andin which usually at least one of the two expected EcoRI restrictionsites at the ends of the sequence previously defined as “spacer region”is absent (Rohde et al., J. Genet. & Breed. 49 (1995), 179-186) from thepreviously identified elements (Rohde et al., J. Genet. & Breed. 46(1992), 391-394). This spacer region shows high homology to thecopia-like BARE-1-element from barley (FIG. 1A; Manninen and Schulman,Plant. Mol. Biol. 22 (1993), 829-846). Thus, the subfamily of copia-likesequences in the coconut genome represent tandem repeats, which displayhomology to the endonuclease and reverse transcriptase RNAse H region ofa copia or copia-like element (see FIG. 1B). The observed sequencedeviations in the elements of the subfamily could now—in contrast to theabove-described attempts for the Ecorep sequences—be used for a geneticanalysis in coconut by appropriate PCR primers. This method for a genomeanalysis in coconut was designated as ISTR (inverse sequence taggedrepeat) analysis.

It was now surprisingly found that this subfamily with its highlyconserved sequence appears to be ubiquitous in the plant and animalkingdom since the use of identical ISTR-primers (see also Table 1),which were developed on the basis of coconut sequences determined by us,obtained high polymorphic DNA fingerprints for other plant species aswell as for animals and humans as well as for microorganisms. In thiscontext, not only a multitude of polymorphic markers can be discoveredwhich segregate in the progeny (“single locus/multiple allele”-markers)but also new polymorphic markers arise (individual specific markers),which, for example, are present neither in the father nor in the motherin control cross-breedings (for example cattle, sheep) and which can bepossibly ascribed to recombination events or to the amplification ofspecific genomic regions. Conclusively, each fingerprint is unique forthe individual progeny from identical parents. In the field of humanbiology it could be demonstrated that this also holds true for identicaltwins for which several of the used ISTR primer pairs displayfingerprints that are different from each other (see FIG. 8).

Thus, a preferred embodiment of the use according to the invention ischaracterized in that with the primer or primer pair a fingerprint isobtainable with DNAs from the entire animal and plant kingdom,comprising

(a) the animal kingdom with all its subkingdoms, preferably Metazoanincluding the subphylum of the vertebrates, preferably the class ofMammals, including in particular the family of the Hominids and thefamily of the Bovidae, including the species Bovis tourus and Ovis ariesas well as all races and varieties which are derivable from thecorresponding species;

(b) the plant kingdom with all its subkingdoms, preferably Mycobiontaand Cormobionta, among the latter preferably the division of theSpermatophyta, therein preferably the class of Monocotyledonae with itsfamilies of the Arecaceae and its representatives of the species Cocosnucifera or the family of Poaceae with its representatives of thespecies Hordeum vulgare and Zea mays, in addition most preferably theclass of the Dicotyledonae with its families, for example Solanaceae andits representatives of the species Solanum tuberosum, Nicotiana tabacum,Petunia hybrida, or e.g., the family of Brassicaceae with itsrepresentatives of the species Brassica napus or the family of theChenopodiaceae with its representative Beta vulgaris or the family ofthe Vitaceae with its representative Vitis vinifera as well as allvarieties and cultivars which are derivable from the correspondingspecies; and

(c) humans; and

(d) microorganisms comprising prokaryotic microorganisms, preferablygram-positive bacteria such as, e.g., lactic acid bacteria, sarcina andcoryneform bacteria and gram-negative bacteria such as, e.g., Neisseriaand enterobacteria, and eukaryotic microorganisms comprising fungi,preferably Phycomycetes such as, e.g., Phytophthora and Ascomycetes suchas, e.g., yeasts.

A particular advantage of the use according to the present invention isthat fingerprints of comparable resolution and sensitivity can bevisualized with DIG labeled PCR products directly in the gel without thegenerally used transfer of the DNA fragments onto membranes (Southernblot) well-known in the art. Thus, the present invention allows toprepare such fingerprints in a simple way (separation of the PCRfragments in a sequence gel, direct detection in the gel, computer-aideddata analysis by directly scanning the sequence gel) without the use ofradioactivity.

Thus, a further preferred embodiment of the use according to theinvention is characterized in that the DNAs to be analyzed is amplifiedwith the primer or primer pair via PCR and subsequently separated on agel according to the length of the PCR products.

From the prior art the person skilled in the art knows how to choose theconditions for an appropriate PCR. Also a method for the separation ofPCR amplified DNAs on an electrophoresis gel which preferably is apolyacrylamide gel is known from the prior art.

In a particularly preferred embodiment, the gel is a sequencing gel. Thepreparation of sequencing gels is also well-known in the art and, forexample, described in Sambrook et al., “Molecular Cloning, A LaboratoryHandbook”. CSH Press, Cold Spring Harbor, 1989.

In a further preferred embodiment, the use according to the invention ischaracterized in that in a further step a Southern blot is performed andthe DNAs transferred onto the membrane are visualized by hybridizationwith a probe.

This embodiment is an alternative to the above-described embodiments. Itrequires more time and/or money and the handling of radioactivity,however, it is perfectly suitable for laboratories which have a lesselaborate lab equipment, for example have no scanner with a connectedcomputer. The performance of Southern blots as well as hybridizationswith an appropriate probe are also well-known in the art and are, forexample, described in Sambrook et al., loc. cit.

In a further particularly preferred embodiment of the use according tothe invention, the probe is the primer or the primer pair of theinvention.

Since the primers are part of the amplified DNA, the detection of thebands on the membrane used for Southern Blot can be easily performed.

In a further preferred embodiment of the use according to the invention,the primer or primer pair is labeled.

In a particularly preferred embodiment of the invention, the label is anon-radioactive label, in particular digoxigenin, biotin andfluorescence dye, a dye or a radioactive label, in particular ³²P.

In particular, the labeling of the primers with digoxigenin and thedyeing of the DNA directly in the gel after amplification and gelelectrophoretic separation of the DNA can be performed by alllaboratories or interested breeders on the basis of a low budgetequipment (PCR reaction, electrophoresis on sequence gels) and withoutthe use of radioactivity. Storage and processing of the data ispreferably performed by direct reading of the dyed and dried gel by ascanner into a computer. Furthermore, the possibility exists to developspecific primers to obtain allele specific amplification products byre-isolation of the PCR products of the sequencing gel and theirre-amplification and sequencing.

In a further preferred embodiment of the invention, the primer displaysthe sequence as depicted in Table 1.

These primers are preferred examples of primers which were used by theinventors in previous fingerprint analyses. It should, however, bepointed out that also other primers can be used which hybridize to thesequence schematically represented in FIG. 2B and which is described inmore detail in Rohde et al., 1992 loc. cit., and Rohde et al., 1995 loc.cit. Moreover, according to the invention, it surprisingly turned outthat all previously tested primers which hybridize to this region canproduce a reliable fingerprint in plants as well as in animals as wellas in humans as well as in microorganisms.

In a further preferred embodiment, the use according to the invention ischaracterized in that the fingerprint analysis is used for studyingbiodiversity, genetic relatedness, taxonomy, and, in particular, in thefield of forensic medicine, breeding, protection of plant varieties,gene library management, population genetics and for studies on theevolution.

Finally, the invention relates to primers for the use according to theinvention, characterized in that the primers display any one of thesequences represented in Table 1.

The Figures show:

FIG. 1: Region of a copia-like element Bare-1 present in the genome ofbarley (FIG. 1A, from Manninen and Schulman, Plant. Mol. Biol. 22(1993), 829-846) which was found as a tandem repeat copia-like sequence(Rohde et al., J. Genet. & Breed. 49 (1995), 179-186) in the genome ofcoconut (Cocos nucifera L.) (FIG. 1B).

(A) Diagram of the copia-like BARE-1 element from barley. ED:Endonuclease; RT: Reverse transcriptase; RH: RNAse H.

(B) Location of repetitive copia-like sequences from coconut relative tohomologous sequences of the barley BARE-1 element. The hatched regioncharacterizes the position of the recently found “spacer region” (Rohdeet al., J. Genet. & Breed. 49 (1995) 179-186).

FIG. 2: Amplification of the “spacer region” between adjacent copia-likesequences in the coconut genome (A) and approximate position ofpreviously used primers for the ISTR analysis (B).

(A) For the amplification for cloning and sequencing of the regionsbetween two adjacent copia-like elements in the coconut the primer pairsISTR5/ISTR-1 (SEQ ID NOS: 5 and 9) and ISTR5/ISTR-2 (SEQ ID NOS: 5 and10) were used. The direction of arrow heads designates the 5′→3′orientation of the oligodeoxynucleotides used.

(B) Usually, each primer is between 18 and 20 nucleotides in length andwas synthesized in analogy to the sequence of the Ecorep1 element (Rohdeet al., J. Genet. & Breed. 49 (1995) 179-186). The primers provided with“-” are complementary to the coding sequence of the copia element andcan be combined with any primer of the “plus” series for the ISTRanalysis.

FIG. 3: ISTR analysis of populations exemplified by coconut (from Rohdeet al., J. Genet. & Breed. 49 (1995) 179-186).

(A) In lanes 1 to 7, single palm trees of an East African Tall (EAT)population were characterized by ISTR analysis with primer pairsISTR5/ISTR-2 (SEQ ID NOS: 5 and 10) (left) and ISTR5/ISTR-1 (SEQ ID NOS:5 and 9) (right), respectively. Lanes 8 and 9 are control analyses of asingle Rennell Island Tall (RLT)- or Pemba Red Dwarf (PRD)-palm tree.

(B) ISTR analysis of two Malayan Yellow Dwarf (MYD)-populations fromTanzania and the Philippines with the primer pair ISTR5/lSTR-2(SEQ IDNOS: 5 and 10).

FIG. 4: General application of ISTR primers in the plant kingdom.

DNA of different plant species was subjected to amplification withprimers ISTR5/ISTR-2(SEQ ID NOS: 5 and 10). The ISTR products in theseparate lanes correspond to the following plants:

1: tobacco, 2: barley, 3: potato, 4: maize, 5: snap dragon, 6:Arabidopsis, 7: rape seed, 8: Craterostigma, 9: petunia, 10: parsley,11: sisal, 12: Milala palm, 13: Borassus palm, 14: coconut palm, 15:sugar beet, 16: Cuphea, 17: yeast.

FIG. 5: ISTR analysis of individual members of the Arecaceae (Palmae).DNAs of 17 different palm trees were subjected to a standard PCRreaction with primers. ISTR5/ISTR-2 (SEQ ID NOS: 5 and 10) and separatedon a 4% PAGE gel. The PCR products in each lane correspond to thefollowing plants:

1: Hyphaene petersiana Mart., 2: Bismarckia nobilis Hildebrandt & H.Wendl., 3: Eugeissona utilis Becc., 4: Korthalsia echinometra Becc., 5:Mauritiella aculeata (H. B. & K.) Burret, 6: Nypa fruticans Wurmb., 7:Pseudophoenix sargentii H. Wendl. ex Sarg., 8: Oraniopsis appendiculata(F.M. Bailey) J. Dransf., Irvine and N. W. Uhl, 9: Socratea exorhizza(Mart.) H. Wendl., 10: Halmoorea tripatha J. Dransf. & N. W. Uhl., 11:Cyrtostachys peekeliana Becc., 12: Deckenia nobilis H. Wendl., 13:Oncosperma tigillarium (Jack) Ridley, 14: Syagrus amara (Jacq.f.) Mart.,15: Attalea allenii H. E. Moore ex L. H. Bailey, 16: Scheelea insignis(Mart.) Karsten, 17: Asterogyne martiana (H. Wendl.) H. Wendl. exHemsley.

FIG. 6: ISTR analysis of barley varieties.

DNAs of 35 different barley genotypes were amplified in a standard PCRreaction with primers ISTR5/ISTR-2 (SEQ ID NOS: 5 and 10) and the PCRproduct was separated on a 4% PAGE gel. In the individual lanes the PCRproducts of the following plants were applied:

1: Fiction, 2: Kaskade, 3: Red, 4: Georgie, 5: Alexis, 6: Marinka, 7:Flash, 8: Portikos, 9: Aura, 10: Gimpel, 11: Prisma, 12: Gitane, 13:Gavotte, 14: Manila, 15: Pilastro, 16: Masto, 17: Torrent, 17: Torrent,18: Thibault, 19: Onice, 20: Mette, 21: Robur, 22: Probidor, 23: Tania,24: Mario Otter, 25: Nico, 26: Magie, 27: Vogelsanger Gold, 28: Tekto2002, 29: Asse, 30: Calcaroides-C15 (ex Bonus), 31: calcaroides-b2 (exBonus), 32: calcaroides-b19 (ex Bonus), 33: Bonus, 34: Christina, 35:Nudinka.

FIG. 7: Analysis of a cattle family (A) and two sheep families (B, C).

(A) Five offspring as well as both mother and father of 3 cattle familywere subjected to ISTR analysis with the primer pair ISTR5/ISTR-2(SEQ IDNOS: 5 and 10). V: Father, M: Mother. The individual offspring arenumbered. The arrow points out a marker which is not present in allindividuals of the offspring.

(B, C) Analysis of two sheep families with offspring of a cross-breedbetween the identical father and mother M1 (B) as well as mother M2

(C). Arrows indicate segregating ISTR markers; asterisks point toindividual-specific markers, which are present neither in the parentsnor in the brothers and sisters.

GSM: Marker (lower band of the triplet), which cosegrates with the malesex. V: Father. The individual offspring of the different breedings arenumbered.

FIG. 8: Analysis of three human families I, II and III with differentprimer pairs.

(A) ISTR analysis with the primer pair ISTR6/ISTR-1(SEQ ID NOS: 6 and9).

(B) ISTR analysis with the primer pair ISTR6/ISTR-2(SEQ ID NOS: 6 and9).

V: Father of offspring; M: Mother; SSM: sex-specific marker. Theoffspring are numbered. The two offspring of families I and 11 areidentical twins.

FIG. 9: FIG. 9 is a DNA analysis of grape varieties; for ISTRfingerprint analysis DNA from 19 different grape genotypes was subjectedto a PCR reaction with the primer pair ISTR5/ISTR-2(SEQ ID NOS: 5 and10). In the individual lanes the PCR products of the following plantswere applied:

1: Sangiovese piccolo precoce, 2: Sangiovese dell'Elba, 3: Sangiovesepolveroso Bonechi, 4: Colorino americano, 5: Prugnolino medio, 6:Colorino del Valdarno, 7: Morellino, 8: Brunellone, 9: Sangiovese forte,10: Sangiovese R10, 11: Saragiolo, 12: Colorino di Pisa, 13: Prugnolinodoice, 14: Morellino di Scansano, 15: Colorino di Lucca, 16: Giacche,17: Tinturier, 18: Sangiovese polveroso, 19: Prugnolo gentile.

FIGS. 10A, B, C: Analysis of Phytophthora palmivora isolates from thePhilippines with the primer combination ISTR5/ISTR-2(SEQ ID NOS: 5 and10)

1: #P8704 (DRCO89; Davao City, Mindanao); 2: #P8646 (DRC001; Davao Sur,Mindanao); 3: #P8652 (DRC007; Davao City, Mindanao); 4: #P8650 (DRC005;Davao City, Mindanao); 5: #P8698 (DRC082; Zamboanga, Mindanao); 6:#P8684 (DRC065; De Oro City, Mindanao); 7: #P8676 (DRC053; Davao City,Mindanao); 8: #P8653 (DRC008; Davao Norte, Mindanao); 9: #P8647 (DRC002;Davao Norte, Mindanao); 10: #P8649 (DRC004; Davao Norte, Mindanao); 11:#P8662; 12: #P8663 (DRC030; Davao Norte, Mindanao); 13: #P8667 (DRC036;South Cotabato, Mindanao); 14: #P8651 (DRC006; Davao Sur, Mindanao); 15:#P8674 (DRC047; Batangas, Luzon); 16: #P8660 (DRC025; Laguna, Luzon);17: #P8705 (DRC090; Davao Norte, Mindanao); 18: #P8665 (DRC033; SouthCotabato, Mindanao).

M: control reaction with DNA of MRD (Malayan Red Dwarf) coconut palm.

The examples explain the invention.

EXAMPLE 1

Detection of Length Polymorphism in the Coconut

For this experiment as represented in FIG. 3, primer pairs ISTR5/ISTR-2(SEQ ID NOS: 5 and 10) and ISTR5/ISTR-1 (SEQ ID NOS: 5 and 9) (seeTable 1) were used. The DNAs to be analyzed were obtained of single palmtrees of populations from East African Tall (EAT) and Malayan YellowDwarf (MYD) as well as from a single palm tree Rennel Island Tall (RLT)and Pemba Red Dwarf (PRD). The oligodeoxynucleotides employed (primers)were ³²p radioactively labeled at their ends via polynucleotide kinaseby known means and subjected to a PCR reaction. This was conventionallyperformed in a volume of 20 μl and contained 1 pmol of each of theprimers and 25 ng of the genomic DNA to be amplified in 1×PCR reactionbuffer (e.g. of the company GIBCO/BRL), 2.5 mM MgCl₂, 0.25 mM dNTP(deoxynucleoside triphosphate), and 1 unit Taq DNA polymerase. First themixture is subjected for three minutes to 95° C. for denaturationfollowed by 40 cycles of 95° C. (30 seconds, denaturing), 45° C. (30seconds, annealing) and 72° C. (2 minutes, synthesis). The reaction endswith a step at 72° C. for ten minutes (synthesis), 10 μl of a dyemixture (in formamide) are added and after heating 3 μl thereof areseparated on a 4% polyacrylamide sequencing gel. After separation of theglass plates, the gel is dried in a known manner on one of thesequencing plates and the separated radioactively labeled PCR productsare made visible by exposure to an X-ray film. This experimentalprotocol applies also to all other ISTR primers and to the followingexamples.

As can be inferred from FIG. 3, some of the DNA products are common toall palms but also differences in the individual palm trees of bothpopulations are observed. This is not surprising for the “Tall” type EAT(FIG. 3A), since for this coconut type cross fertilization in the fieldhas been observed. However, surprisingly, the ISTR analysis discoversalso differences in the “dwarf” palm type such as MYD which was commonlysupposed to be autogamous within the populations as well as differencesbetween the populations from Tanzania and the Philippines (FIG. 3B).Such differences in dwarf populations could not be detected withpreviously used RFLP markers. Furthermore, it appears that the use ofthe primer pair ISTR5/ISTR-1 (SEQ ID NOS: 5 and 9) not only - asexpected from the position of the ISTR-1 (SEQ ID NO; 9) primer (FIG.2B)—generates PCR products which are approximately 100 base pairsshorter in length but also causes new polymorphisms. The reason for thiscan only be speculated on but the finding opens the possibility of usingall conceivable copia-like sequences and primer combinations for theISTR analysis based on the ascertained copia-like sequences in thecoconut Thus, this simple experiment impressively demonstrates how asingle PCR amplification using the identical primer pair allows areproducible fingerprint analysis of individual palm trees andstatements to the genetic homogeneity of populations.

EXAMPLE 2

Test for a General Application in Plants

In order to find out whether it is generally possible to use thecoconut-specific ISTR primers for the detection of DNA polymorphisms incopia-like sequences in plants, for the experiment of Example 2, thegenomic DNAs of different plants were subjected to PCR reaction with theISTR primer pair ISTR5/ISTR-2(SEQ ID NOS: 5 and 10). From FIG. 4 it canbe seen that from tobacco to yeast DNA all DNAs give individual PCRproducts by use of the coconut-specific primers. Similar experimentswere also performed with other ISTR primer combinations. This shows thatfamilies of adjacent copia-like repetitive elements similar to thosedescribed for coconut exist in lower and higher plants and areaccessible for fingerprint analysis. As a consequence, the ISTR analysisis not only applicable for a single plant species as described inExample 1 but also for the characterization of genetic diversity and theassessment of plant genetic resources either in gene libraries or byin-situ conservation.

EXAMPLE 3

Test for the Application within a Plant Family Exemplified for Palms(Arecaceae)

The possible application of the ISTR analysis for taxonomic studies wasperformed with the ISTR primer pair ISTR5/ISTR-2 (SEQ ID NOS: 5 and 10)in plant species of the family Arecaceae (Palmae). In this experiment,DNAs of 17 palm species (see legend of FIG. 5) were amplified in a PCRreaction with the mentioned primers and PCR products were analyzed in aknown manner. As can be seen in FIG. 5, for each palm a differentfingerprint is obtained, which permits the processing of the data viacomputer-aided evaluation of a corresponding matrix for the assessmentof biological diversity by means of generation of dendrograms accordingto conventional methods. Important for the practical usability is, forexample, which genetic relatedness exists, e.g. between the importantoil plants of the oil and the coconut palm. Genetic markers, forexample, for the feature of the thickness of the nutshell, which isimportant for the yield of oil, could be used in both species forbreeding purposes if they are genetically highly related.

EXAMPLE 4

Test for Application in Highly Cultivated Varieties Illustrated forBarley

Characterization of cultivated varieties via fingerprint analysis bymeans of the ISTR technology was tested by the example of barleyvarieties. FIG. 6 shows a PAGE analysis of PCR products which wereobtained for a total of 35 varieties and/or genotypes. The high geneticrelatedness of the highly cultivated barley varieties investigated isapparent from the high number of monomorphic DNA fragments. However,even in this single analysis a total of 44 polymorphic markers could beidentified which were mainly located in the upper part of the sequencinggel. These markers were grouped in a matrix and based on the matrix adendrogram was evaluated by the UPGMA method. The fact that the varietyBonus (lane 33) could not be distinguished from calcaroides-b19 (lane32) is not surprising since this genotype is a recessive mutant ofBonus. This, however, holds also true for the genotypes Calcaroides-C15(lane 30) and calcaroides-b2 (lane 31) which were generated viamutagenesis in the same genetic background. However, in this case,neutron radiation (Calcaroides-C15) and X-radiation (calcaroides-b2)were used as mutagenes, which usually lead to deletions and inversionson the chromosomal level, while calcaroides-b19 was obtained from Bonusvia sodium azide treatment, which causes point mutations. Firstly, thisexample thus illustrates that the ISTR analysis is suitable to giveindications of rearrangements of the genetic material. Secondly, the useof a single ISTR primer pair is sufficient for a fingerprint of highlycultivated varieties. Thus, it can be concluded that by using furtherISTR primer pairs an unambiguous variety-specific fingerprint can beobtained, which serves for biochemical characterization of the variety(protection of plant varieties).

EXAMPLE 5

Test for Application in Animals: Evidence for Segregating and NewlyDeveloping Markers in Families

In order to test general applicability of the ISTR analysis for geneticmaterial outside the plant kingdom, animal families were investigated inwhich the father was known because of controlled breeding (in-vitrofertilization). FIG. 7 illustrates an ISTR analysis with the primer pairISTR5/ISTR-2 (SEQ ID NOS: 6 and 9) of a cattle family (FIG. 7A) and oftwo sheep families with identical father but two different mothers M1(FIG. 7B) and M2 (FIG. 7C). Both analyses reveal that 1) coconutspecific ISTR primers can also be used in the animal kingdom forfingerprint analysis and that 2) segregating markers (see arrows in FIG.7C) as well as individual specific markers (see asterisks in FIG. 7) areaccessible via ISTR analysis. An indication that segregating ISTRmarkers are capable of cosegregating with important phenotypes isevidenced by the DNA band of the prominent triplet designated as SSM(sex specific marker) in FIGS. 7B, C: This band is present in the fatherbut not in the two mothers. In fact, both of the offspring of family 1(FIG. 7B) are female while family 2 (FIG. 7C) has a male progeny. Thefacts that the parental markers are not present in all offspring (seearrow in FIG. 7A) and that new markers developed (see asterisks in FIG.7), can be interpreted as an indication that the ISTR analysis iscapable of discovering recombination events in cross-breedings.

EXAMPLE 6

Test for Application in Humans: Evidence for Sex and Individual-specificPolymorphisms

This example illustrates the application of the ISTR analysis in thefield of humans. For humans, three families I, II and III were analyzedwherein both children of families I and II were homozygous (identical)twins. Since it could not be expected that ISTR primers were capable ofdiscovering DNA polymorphisms in identical twins (highly polymorphicmicrosatellite primers do not display any differences, Haas, Institutfür Rechtsmedizin, Universitat Giessen; personal communication), 6different ISTR primers were tested. In all 6 analyses DNA polymorphismsare visible, and two of the ISTR analyses of the primer pairsISTR6/ISTR-1 (SEQ ID NOS: 6 and 9) and ISTR6/ISTR-2 (SEQ ID NOS: 6 and10) are shown in FIG. 8. The analysis with the primer pair ISTR6/ISTR-1(SEQ ID NOS: 6 and 9) (FIG. 8A) is remarkable for the multitude ofpolymorphic DNA bands which are individual-specific and provide anunambiguous characterization of the individual human even in the twopairs of identical twins of families I and II. This also holds true forthe ISTR analysis performed with the primer pair ISTR6/ISTR-2 (SEQ IDNOS: 6 and 10) shown in FIG. 8B, although the number of polymorphicbands is lower. Most remarkably, one DNA band is found among the newpolymorphisms (SSM in FIG. 8B) which is only present in the threefathers but not in the three mothers or in the five children. Actually,it is possible that, as mentioned in Example 5 for the sheep families,said DNA band concerns a sex-specific marker since all five children arefemale and thus a strictly sex-specific segregation within a total of 11individuals is given.

EXAMPLE 7

Detection of ISTR Fingerprints for Grape Varieties

For this test, which is represented in FIG. 9, the primer pairISTR5/ISTR-2 (SEQ ID NOS: 5 and 10) (see Table 1) was used. As DNAs tobe examined the genomic DNAs of 19 Vitis vinifera L. plants as well as13 suspected “Sangiovese” genotypes and 6 “colored” ecotypes were used,the fruit of which is of importance for the intensive red coloration ofthe wine. It is evident from FIG. 9 that a large number of polymorphousDNA fragments was obtained. Although the variability is highest in the“colored” ecotype, ISTR analysis evidenced also a high proportion ofpolymorphisms in the “Sangiovese” genotypes. These differences canpossibly be ascribed to the polyclonal origin of many grape cultivars.Therefore, this example, too, proves that ISTR analysis is an efficientand sensitive method for examining the genetic diversity within ecotypesand for the identification of individual clones.

EXAMPLE 8

Use of the ISTR Fingerprints in Microorganisms

The use of the ISTR technique in microorganisms was exemplified forisolates of the fungus Phytophthora palmivora which induces lethaldiseases (“bud rot”) in coconut palms. A particularly difficult examplewas chosen for the use of a DNA marker technology for which, due to thelimited genetic diversity, originally only few polymorphisms wereexpected since in all cases P. palmivora isolates were used which weremoreover exclusively isolated in the Philippines and were locallylimited (the isolates were mainly derived from Mindanao island).

1 μg DNA each was amplified of eighteen P. palmivora isolates from thePhilippines in a standard PCR reaction with the primer combinationISTR5/ISTR-2(SEQ ID NOS: 5 and 10), the products were separated by PAGEon a 4% polyacrylamide gel in a conventional manner and the individualbands were visualized by autoradiography. FIG. 10 shows the result ofthis analysis. Already the gel analysis (FIG. 10A) yields a largevariety of polymorphous DNA fragments with a single ISTR primercombination. Thirty of these bands were analyzed according to the knownmethod of the cluster analysis to phenograms according to the UPGMAmethod (SAHN clustering; FIG. 10B) and by PCA (principal coordinateanalysis; FIG. 10C). The data obtained corresponded well to theclassification of these isolates using the RAPD-DNA marker analysis.

TABLE 1 Examples of oligodeoxynucleotides (ISTR primer) used for theISTR analysis ISTR primer sequence (5′→3′) Forward primer ISTR1 SEQ IDNO:1 AGG AGG TGA ATA CCT TAG ISTR2 SEQ ID NO:2 AAA ATG GCA TAG TCT CTCISTR3 SEQ ID NO:3 GTC GAC ATG CCA TCT TTC ISTR4 SEQ ID NO:4 TAT AGT ACCTAT TGG GTG ISTR5 SEQ ID NO:5 ATA TAT GGA CTT AAG CAA GC ISTR6 SEQ IDNO:6 GTA TTG TAC GTG GAT GAC ATC ISTR7 SEQ ID NO:7 CAA CAG TGC TCC CACTGA ISTR7 SEQ ID NO:8 TGC TAG GAC TTT CAC AGA Backward primer ISTR-1 SEQID NO:9 TTT TCT ACT TCA TGT CTG A ISTR-2 SEQ ID NO:10 AAT AAA TCG ATCATC GAC ISTR-3 SEQ ID NO:11 ATT CCC ATC TGC ACC AAT ISTR-4 SEQ ID NO:12ATG TCA TCC ACG TAC AAT ISTR-5 SEQ ID NO:13 CTT CTG TGA AAG TCC TAG

13 1 18 DNA Artificial Sequence Description of Artificial Sequenceprimerthat hybridizes to copia-like sequences 1 aggaggtgaa taccttag 18 2 18DNA Artificial Sequence Description of Artificial Sequenceprimer thathybridizes to copia-like sequences 2 aaaatggcat agtctctc 18 3 18 DNAArtificial Sequence Description of Artificial Sequenceprimer thathybridizes to copia-like sequences 3 gtcgacatgc catctttc 18 4 18 DNAArtificial Sequence Description of Artificial Sequenceprimer thathybridizes to copia-like sequences 4 tatagtacct attgggtg 18 5 20 DNAArtificial Sequence Description of Artificial Sequenceprimer thathybridizes to copia-like sequences 5 atatatggac ttaagcaagc 20 6 21 DNAArtificial Sequence Description of Artificial Sequenceprimer thathybridizes to copia-like sequences 6 gtattgtacg tggatgacat c 21 7 18 DNAArtificial Sequence Description of Artificial Sequenceprimer thathybridizes to copia-like sequences 7 caacagtgct cccactga 18 8 18 DNAArtificial Sequence Description of Artificial Sequenceprimer thathybridizes to copia-like sequences 8 tgctaggact ttcacaga 18 9 19 DNAArtificial Sequence Description of Artificial Sequenceprimer thathybridizes to copia-like sequences 9 ttttctactt catgtctga 19 10 18 DNAArtificial Sequence Description of Artificial Sequenceprimer thathybridizes to copia-like sequences 10 aataaatcga tcatcgac 18 11 18 DNAArtificial Sequence Description of Artificial Sequenceprimer thathybridizes to copia-like sequences 11 attcccatct gcaccaat 18 12 18 DNAArtificial Sequence Description of Artificial Sequenceprimer thathybridizes to copia-like sequences 12 atgtcatcca cgtacaat 18 13 18 DNAArtificial Sequence Description of Artificial Sequenceprimer thathybridizes to copia-like sequences 13 cttctgtgaa agtcctag 18

What is claimed is:
 1. A method for DNA fingerprint analysis consistingessentially of annealing a single primer consisting of 15-25 contiguousnucleotides of the complement of a 1.4 kilobasepair copia-like elementof the DNA of Cocos nucifera L. to a sample of genomic DNA, obtaining aplurality of polymerase chain reaction products products by performing apolymerase chain reaction using said single primer and obtaining a DNAfingerprint by separating the polymerase chain reaction products.
 2. Themethod of claim 1, wherein said genomic DNA is obtained from a human, anon-human animal, a plant or a microorganism.
 3. The method of claim 1,wherein said genomic DNA is obtained from a human, a non-human animal ora plant.
 4. The method of claim 1, wherein said genomic DNA is obtainedfrom an organism that is a mammal, a monocotyledenous plant or adicotyledenous plant.
 5. The method of claim 1, wherein said genomic DNAis obtained from an organism of the family Hominidae, Bovidae, Arecaceaeor Poaceae.
 6. The method of claim 1, wherein said genomic DNA isobtained from an organism of the genus Homo, Bovis, Ovis, Hordum, Zea,Solanum, Nicotiana, Petunia, Brassica, Beta, Vitus or Neisseria.
 7. AThe method of claim 1, wherein said genomic DNA is obtained from afungus, a lactic acid bacterium, a sarcina bacterium, a coryneformbacterium or a gram-negative bacterium.
 8. The method of claim 1,wherein said genomic DNA is obtained from a fungus that is of the familyPhytophthora or Ascomycetes.
 9. The method of claim 1, wherein saidprimer has a nucleotide sequence of any one of SEQ. ID. NOS: 1 to 13.10. The method of claim 1, wherein said separating step comprisesseparating said polymerase chain reaction products on a sequencing gel.11. The method of claim 1, further comprising Southern blotting saidpolymerase chain reaction products.
 12. The method of claim 11, whereinsaid Southern blotting is performed using as a probe a labeledoligonucleotide having a nucleic acid sequence of any one of SEQ. ID.NOS.: 1 to
 13. 13. The method of claim 12, wherein said label isdigoxigenin, biotin, a fluorescent dye or a radioactive label.
 14. Themethod of claim 1, wherein said copia-like element comprises a Sal Irestriction site and a Sca I restriction site.
 15. The method of claim1, wherein said primer is from 15 to 25 nucleotides in length.
 16. Amethod for DNA fingerprint analysis comprising annealing at least oneprimer consisting of 15-25 contiguous nucleotides of the complement of a1.4 kilobasepair copia-like element of the DNA of Cocos nucifera L. to asample of genomic DNA, wherein said sample of genomic DNA is obtainedfrom an organism other than a plant of the genus Cocos, obtaining aplurality of polymerase chain reaction products by performing apolymerase chain reaction and obtaining a DNA fingerprint by separatingthe polymerase chain reaction products.
 17. The method according toclaim 16, wherein a pair of primers is utilized in said polymerase chainreaction.
 18. The method of claim 16, wherein said at least one primeris from 15 to 25 nucleotides in length.
 19. The method of claim 17,wherein said pair of primers is SEQ ID NOS: 5 and
 9. 20. The method ofclaim 17, wherein said pair of primers is SEQ ID NOS: 5 and
 10. 21. Themethod of claim 17, wherein said pair of primers is SEQ ID NOS: 6 and 9.22. The method of claim 17, wherein said pair of primers is SEQ ID NOS:6 and
 10. 23. The method of claim 17, wherein said pair of primersconsists of a first primer selected from the group consisting of SEQ IDNOS: 1-7 and a second primer selected from the group consisting of SEQID NOS: 9-13.
 24. The method of claim 16, wherein said genomic DNAsample is obtained from a human, a non-human animal or a microorganism.25. The method of claim 16, wherein said organism is a monocotyledenousplant.
 26. The method of claim 16, wherein said genomic DNA is obtainedfrom an organism of the family Hominidae, Bovidae, Arecaceae or Poaceae.27. The method of claim 16, wherein said genomic DNA is obtained from anorganism of the genus Homo, Bovis, Ovis, Hordum, Zea, Solanum,Nicotiana, Petunia, Brassica, Beta, Vitus or Neisseria.
 28. The methodof claim 16, wherein said genomic DNA is obtained from a fungus, alactic acid bacterium, a sarcina bacterium, a coryneform bacterium or agram-negative bacterium.
 29. The method of claim 16, wherein saidgenomic DNA is obtained from a fungus that is of the family Phytophthoraor Ascomycetes.
 30. The method of claim 16, wherein each of said atleast one primer has a nucleotide sequence of any one of SEQ. ID. NOS.:1 to
 13. 31. The method of claim 16, wherein said separating stepcomprises separating said polymerase chain reaction products on asequencing gel.
 32. The method of claim 16, further comprising Southernblotting said polymerase chain reaction products.
 33. The method ofclaim 32, wherein said Southern blotting is performed using as a probe alabeled oligonucleotide having a nucleic acid sequence of any one ofSEQ. ID. NOS.: 1 to
 13. 34. The method of claim 33, wherein said labelis digoxigenin, biotin, a fluorescent dye or a radioactive label. 35.The method of claim 16, wherein said copia-like element comprises a SalI restriction site and a Sca I restriction site.
 36. A primer pairselected from the group consisting of SEQ ID NOS:5 and 10, SEQ ID NOS: 6and 9, and SEQ ID NOS:6 and 10.